1. Field of the Invention:
The present invention relates to a method for rapidly determining the effectiveness of antimicrobial agents against bacteria. More particularly, the invention relates to a method for rapidly determining the effectiveness of antimicrobial agents against bacteria by measuring the amount of adenosine triphosphate (ATI) isolated from the bacteria remaining after treatment with the antimicrobial agent.
2. Description of the Prior Art:
A rapid and routine procedure for the determination of the effectiveness of antimicrobial agents against various bacteria is very frequently of vital importance, particularly for the rapid measurement of the effectiveness of antimicrobial agents against bacteria in urine. Present techniques for the determination of microbial susceptibility generally require overnight incubation of an infecting organism after its isolation.
Thus, generally a minimum of 48 hours is required to test the effectiveness of antimicrobial agents after a specimen is received in the laboratory. The most commonly used conventional techniques for the determination of microbial susceptibility to antimicrobial agents include agar diffusion (Kirby-Bauer technique), broth dilution and agar dilution. A long-standing need continues to exist for a rapid means for determining microbial sensitivity to antimicrobial agents to avoid the inclusion of inappropriate, unnecessary, and often toxic agents in the therapeutic regimen of an infected person.
The conventional broth dilution method (MIC-Broth Dilution) of determining microbial sensitivity to antimicrobial agents involves visual detection of the least amount of antimicrobial agent required to cause complete inhibition of bacterial growth in a culture medium. In the technique, two-fold dilutions of antibiotics are made in a suitable culture medium. A control tube containing the culture medium, but no antibiotic is also included for each organism tested. The organism is then allowed to grow to a logarithmic or early stationary phase of growth in the medium, and then diluted to a solution containing 10.sup.4 to 10.sup.5 viable units per milliliter. A quantity of the cultured medium is then placed in each tube of a series containing varying amounts of an antibiotic, and the tubes are allowed to incubate at 37.degree. C for 16 to 20 hours. Thereafter, the end point of the test is determined visually, as mentioned above. A disadvantage of this technique besides the relatively very long time required to complete the test, is that care must be taken to recognize that slight amounts of turbidity present may be caused by the inoculum itself and not by the growth of the organism.
The Kirby-Bauer method involves an agar diffusion technique for the measurement of microbial susceptibilities. In the test an inoculum of an organism, which is an overnight culture, is prepared by diluting the inoculum in trypticase soy broth to such a concentration that a dense, but not confluent growth is observed. The culture turbidity is adjusted to a concentration which conforms to a turbidity standard. The standard is formed by mixing 0.5 ml of 0.048 M barium chloride (1.175% w/v Ba Cl .sup.. 2H.sub.2 O) with 99.5 ml of 0.36 NH.sub.2 SO.sub.4 (1% w/v). A cotton swab is then soaked in the diluted culture, and an agar plate is then streaked in four directions to obtain an even and thorough distribution of the organism on the plate. The treated plates are dried for 15 minutes at 37.degree. C. Thereafter, antibiotic discs are applied to the surface of the agar and pressed into place. No more than 12 discs are used per plate to prevent overlapping of zones. The plates are then allowed to stand at room temperature for 30 minutes and incubated at 37.degree. C for 18 to 20 hours. The end point is shown by a clearly defined zone around the antibiotic disc where no growth has occurred. The zone is measured and compared with the given sensitivity or resistance of the antibiotic. Only a zone having a diameter greater than 6.0 mm is significant because the antibiotic discs are 6.0 mm in diameter. The chief disadvantage of the diffusion technique is the long period of time required to obtain the microbial sensitivity results.